Interictal plasma glutamate levels are elevated in individuals with episodic and chronic migraine

Glutamate is implicated in migraine pathogenesis including central sensitization and pain transmission. Altered plasma glutamate levels has been noted in migraine. Chronic migraine (CM) presented a higher degree of central sensitization and pain transmission than episodic migraine (EM). However, no study has evaluated plasma glutamate levels separately in EM and CM. This study aimed to assess plasma glutamate levels in EM and CM compared to controls. An enzyme-linked immunosorbent assay was used to assess plasma glutamate levels in females with EM (n = 98) and CM (n = 92) as well as controls (n = 50). Plasma glutamate levels in participants with EM (median and interquartile range, 49.73 [40.82–66.12] μmol/L, p < 0.001) and CM (58.70 [44.64–72.46] μmol/L, p < 0.001) were significantly higher than those in controls (38.79 [29.50–53.60] μmol/L). Glutamate levels were not significantly different between participants with EM and CM (p = 0.075). There was no significant association of plasma glutamate levels with headache frequency (exponential and 95% confidence interval, 1.285 [0.941–1.755]) and intensity (mild, 59.95 [59.95–59.95] μmol/L vs. moderate, 52.76 [40.83–106.89] μmol/L vs. severe, 55.16 [42.34–68.03] μmol/L, p = 0.472). The plasma glutamate level is a potential indicator for EM and CM.

Glutamate is an excitatory neurotransmitter expressed abundantly in humans and is crucially involved in the pathogenesis of migraine, including central sensitization, cortical spreading depression, and trigeminal activation 1 . It is synthesized from glutamine and glucose, and is metabolized into gamma-aminobutyric acid, which is a key inhibitory neurotransmitter 2 . Anti-glutamatergic drugs are used as preventive treatments for migraine 3,4 .
During ictal and interictal periods, individuals with migraine present with altered glutamate levels in body fluids, including plasma, cerebral spinal fluid (CSF), urine, and saliva [5][6][7][8][9][10][11][12] . Many studies have noted elevated plasma glutamate levels during the interictal period. Furthermore, a meta-analysis using data pooled from previous studies found a significant elevation in interictal plasma levels of glutamate in individuals with migraine 13 .
Migraine can be categorized as episodic migraine (EM) and chronic migraine (CM) 14 . These migraine subtypes have different prevalence, comorbidities, and treatment response 15 . A previous study reported differences in the biomarkers for EM and CM 13 . Compared with individuals with EM, individuals with CM reported a higher degree of central sensitization and pain transmission 16 . Considering that glutamate plays a key role in central sensitization and pain transmission, there is a possibility that the plasma glutamate levels in CM differ from those in EM 1 . However, to our knowledge, no study has evaluated plasma glutamate levels separately in individuals with EM and CM.
We hypothesized that compared with healthy controls, individuals with EM and CM have increased interictal plasma levels of glutamate. This study aimed to assess plasma glutamate levels in participants with EM and CM and compare them with those in controls. We also aimed to assess the difference in the plasma glutamate levels between EM and CM. Additionally, the present study aimed to evaluate the diagnostic utility of plasma glutamate levels in differentiating participants with EM and CM from healthy controls. Furthermore, we aimed to investigate the relationship of plasma glutamate levels with clinical characteristics and comorbidities. inter-assay and intra-assay accuracies were 4.21% and 4.11% coefficient of variation, respectively. The estimated inter-assay and intra-assay precisions were 5.09% and 6.43% coefficient of variation, respectively. The aforementioned values were calculated by an independent laboratory blinded to the clinical data.
Estimation of the sample size. The sample size was estimated based on a previous study on the interictal level of plasma glutamate in individuals with migraine 7 . The ratio of participants with migraine to controls was set at 2:1. Assuming a 5% significance level and 80% power, the sample sizes of the migraine and control groups were calculated as 89 and 45, respectively. Accordingly, we targeted to enroll 90 participants with each migraine type (EM and CM). Furthermore, we targeted to enroll 45 participants as healthy controls.
Assessment of anxiety, depression, FM, and medication overuse. Anxiety and depression were examined since they are common comorbidities of migraine and are closely associated with the severity of migraine symptoms 18 , Anxiety and depression were evaluated using Generalized Anxiety Disorder-7 (GAD-7) and Patient Health Questionnaire-9 (PHQ-9), respectively 19,20 . A GAD-7 score ≥ 8 and PHQ-9 score ≥ 10 indicated the presence of anxiety and depression, respectively 21,22 . FM was diagnosed according to the 2016 ACR criteria 17 . Anxiety, depression, and FM were evaluated in all participants with EM and CM, and in controls. Medication overuse (MO) was diagnosed as follows, based on the MOH criteria (code 8.2): regular intake of triptans, ergotamine, combination analgesics, and opioids for ≥ 10 days/month or regular intake of non-opioid analgesics on ≥ 15 days/month for > 3 months. For participants using multiple classes of drugs, we applied the criteria for MOH attributed to multiple drug classes not individually overused (code 8.2.6) 14 . The criterion of headache day frequency (≥ 15 days/month) was not applied in the MO diagnosis.
Statistical analysis. Binary and ordinal variables are showed as numbers and percentages. The normality of data was assessed using the Shapiro-Wilk test. Normally and non-normally distributed continuous variables were examined using an independent t-test or analysis of variance and the Mann-Whitney or Kruskal-Wallis tests, respectively. The results of the latter tests are expressed as medians (interquartile ranges [IQR]). Categorical variables were examined using the chi-square test.
The area under the curve (AUC) for each receiver operating characteristic (ROC) curve was obtained to measure the discrimination capacity of plasma glutamate levels (control vs. EM and control vs. CM). Poisson regression analysis with adjustment for age was used to evaluate the association between the monthly headache frequency and plasma glutamate levels. Post hoc analyses were conducted using Bonferroni's method.
We set the statistical significance at p < 0.05 (two-tailed). For among-group comparisons of plasma glutamate levels through post hoc analyses, statistical significance was set at p < 0.017 (0.050/3). Except for ROC analysis and sample power calculation, all statistical analyses were conducted using IBM SPSS software version 25 for Windows (IBM Corp., Armonk, NY, USA). ROC analysis was conducted using EzR version 1.4.5 23 . Sample power was calculated using G*Power version 3.1.9.6 24 The analyses were planned before data collection. There were no missing data.

Ethical approval. This study was reviewed and approved by the Institutional Review Board of Severance
Hospital, Yonsei University (approval No. 2018-2711-004). This study was conducted following the principles of the Declaration of Helsinki and its subsequent amendments 25 . All participants provided written informed consent before study participation. Additionally, there were significant differences in glutamate levels between participants with migraine without aura and controls (p < 0.001). However, there was no significant difference in the plasma glutamate levels between participants with aura and controls (p = 0.055) as well as between participants with migraine with and without aura (p = 0.244).
Diagnostic utility of plasma glutamate levels. The maximal Yuden index for differentiating between participants with EM and healthy controls was achieved at 40.10 μmol/L, with an AUC of 0.724 (95% bootstrap confidence interval [CI]: 0.633-0.815) ( Fig. 2A). Based on this threshold, 69.6% and 78.6% of participants with EM and healthy controls, respectively, were correctly assigned. The maximal Yuden index for differentiating between participants with CM and healthy controls was achieved at 43.19 μmol/L, with an AUC of 0.764 (95% bootstrap CI: 0.681-0.847) (Fig. 2B). Based on this threshold, 77.4% and 66.0% of participants with CM and healthy controls, respectively, were correctly assigned. The diagnostic utility at these thresholds for EM and CM including sensitivity, specificity, accuracy, positive predictive value, and negative predictive value were estimated (Table 2). www.nature.com/scientificreports/   Plasma glutamate levels according to preventive medications. Sixty participants (31.6%) with EM or CM received preventive treatments for migraine. None of the participants received anti-calcitonin generelated peptide antibody or botulinum toxin A treatment. Plasma glutamate levels did not significantly differ according to the classes of migraine preventive medications taken by the participants (Table 4). Furthermore, plasma glutamate levels did not significantly differ according to the use of anti-glutamatergic medications (topiramate and zonisamide).

Discussion
Our study presented several major findings. First, compared with the control group, the EM and CM groups showed significantly increased plasma glutamate levels. Second, there was no significant difference in plasma glutamate levels between the EM and CM groups. Third, there was no significant association of glutamate levels with headache frequency, headache intensity, most typical headache characteristics, and preventive treatment. These findings confirm our hypothesis that plasma glutamate levels are elevated in individuals with EM and CM.
Several studies have reported increased ictal and interictal elevation of plasma glutamate 5,[7][8][9]26,27 . However, none of the studies have evaluated plasma glutamate levels separately in participants with EM and CM 13 or the  Table 4. Glutamate levels according to the classes of preventive medications.  www.nature.com/scientificreports/ association of clinical characteristics with glutamate levels. Our findings suggested that the interictal plasma glutamate levels may reflect the presence, rather than status, of migraine. The mechanisms underlying the increased plasma levels in participants with EM and CM remain unclear. One possible explanation is that the increase in glutamate levels in neurons and platelets can affect plasma glutamate levels in participants with migraine. Neurons and platelets are the main sources of plasma glutamate 28,29 . Individuals with migraine show increased interictal CSF glutamate level, which suggests they have elevated glutamate levels in the central nervous system (CNS) 10,12,30,31 . Moreover, neuroimaging studies have demonstrated increased interictal glutamate levels in participants with migraine 32,33 . Although glutamate cannot pass through the blood-brain barrier (BBB), excessive neuronal glutamate shift from the CNS to plasma may occur through increased BBB permeability via activation of the N-Methyl-D-aspartic acid receptor 34 . Furthermore, glutamate transfer can occur in small CNS regions lacking the BBB, which are termed as circumventricular organs 35 . There is a close correlation between plasma and CSF glutamate levels 36 . Moreover, platelets can be a glutamate source since, similar to glutamatergic neurons, they have high-affinity glutamate transporters and contain glutamate granules 37,38 . Platelet glutamate can contribute to glutamate accretion in the brain 29 . Participants with migraine also show increased levels of platelet glutamate 26 .
In our study, the diagnostic accuracy of plasma glutamate levels for differentiating participants with EM and CM from healthy controls was moderate for EM (AUC = 0.724) and CM (AUC = 0.764) 39 . Ideally, we should select tests with high sensitivity, specificity, and accuracy; however, it is also important to consider the characteristics of the conditions being tested 40 . Most biomarker studies on migraine headaches have reported increased plasma glutamate levels 13,41 . However, none of the studies have reported the diagnostic utility of plasma glutamate levels in the diagnosis of EM and CM. Our findings could inform future biomarker studies on migraine.
Increased salivary glutamate levels have been reported in individuals with CM, but not EM 11 . In contrast, we observed no significant differences in glutamate levels between the EM and CM groups. This inconsistency could be attributed to differences in the sample sources. Alterations in glutamate levels in participants with migraine may differ across samples. For example, compared with controls, participants with migraine with and without aura have altered plasma glutamate levels 27 . However, there was no significant among-group difference in the erythrocyte glutamate levels.
There is substantial evidence indicating the role of glutamate in migraine pathogenesis. Genome-wide association studies implicate genes that are involved with glutamate signaling in migraine, and gene mutations responsible for familial hemiplegic migraine and other familial migraine syndromes may influence glutamate signaling 42,43 . Animal studies indicate that glutamate plays a key role in pain transmission, central sensitization, and cortical spreading depression [44][45][46] . Multiple therapies that target glutamate receptors, including magnesium, topiramate, memantine, and ketamine, have been reported to have efficacy in the treatment of migraine 47 . Glutamate contributes to endothelial dysfunction through oxidative stress and apoptosis 48 . This finding suggests a role of glutamate in the link between migraine and stroke. Our study provides additional evidence implicating glutamate in the pathogenesis of migraine, especially CM.
Despite presenting some credible evidence in this research field, our study has several limitations. First, we only enrolled women to avoid the potential effects of sex differences in glutamate levels. Therefore, our findings may not reflect glutamate levels in male participants with EM and CM. There have been inconsistent findings regarding sex differences in plasma glutamate levels. Some studies have reported significant sex differences 49 while other studies have reported no significant differences 50,51 . If there is a difference in the levels of glutamate between women and men, the effects of sex-hormones can be considered. Since sex-hormones fluctuate during menstrual cycles, if there is a difference in the glutamate levels between women and men, the relationship between menstrual cycle and blood sampling time should be considered. Second, the present study did not include participants aged < 19 and > 65 years. Although plasma glutamate levels did not significantly differ according to age, findings in the present study did not reflect glutamate levels in that age group 52 . Therefore, it will be necessary to measure the plasma glutamate levels in different age groups. Third, although we enrolled a sufficient sample size based on a previous study, the sample sizes in some subgroup analyses might have been insufficient. Specifically, all glutamate level differences according to clinical characteristics, comorbidities, and preventive treatment did not have sufficient sample power, suggesting an insufficient sample size. Nevertheless, we presented these results to provide more information on the glutamate levels in participants with EM and CM according to various conditions. Therefore, further studies with a sufficient sample size are needed for comparison of glutamate levels among migraine participants according to these conditions. Fourth, we enrolled participants with EM and CM from a single tertiary-care university hospital. Therefore, our findings may lack generalizability. Future studies on various populations are warranted for more generalizable findings. Fourth, we did not evaluate the migraine duration and could not evaluate the relationship of disease duration with glutamate levels. Disease duration and headache frequency are associated with brain damage in individuals with migraine and may affect brain and plasma glutamate levels 53 . However, we observed no significant association of headache frequency with plasma glutamate levels. Nevertheless, there is a need for further studies to assess the association between disease duration and glutamate levels.

Conclusions
In conclusion, we evaluated plasma glutamate levels in participants with EM and CM. Compared with the control group, the EM and CM groups showed increased plasma glutamate levels. However, there was no significant difference in glutamate levels between the EM and CM groups. There were no significant differences in the glutamate levels according to headache frequency, headache intensity, and probably according to preventive treatments, MO, and FM. Our findings demonstrate that the plasma glutamate level is a potential marker for EM and CM. www.nature.com/scientificreports/

Data availability
The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.